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ÍNDICE
Resumo ...
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Introdução ...
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Objetivos ...
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Resultados e Discussões ...
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Artigo Científico 1 ...
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Artigo Científico 2 ...
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Conclusões ...
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Bibliografia ...
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vRESUMO
Caquexia, presente na maioria dos hospedeiros com câncer, é um estado caracterizado pela perda involuntária de peso. Pacientes com caquexia apresentam expectativa de vida muito reduzida e menor qualidade de vida. Nos pacientes com câncer-caquexia há intensa mobilização de substratos dos tecidos da carcaça do organismo hospedeiro ocorrendo, preferencialmente, depleção de proteína muscular em função da redução da síntese e/ou aumento da degradação protéica no músculo. Este aumento da taxa de proteólise muscular tem como função prover aminoácidos para síntese de glutamina, bem como para a demanda de síntese das células neoplásicas. Trabalhos da literatura mostram que o desenvolvimento de câncer dá-se de forma mais agressiva e severa quanto mais jovem for o paciente. Pacientes acometidos pelo crescimento de neoplasia maligna concomitante à gravidez sofrem da mesma agressividade desta doença, com um agravante maior tratam se de dois pacientes: mãe e feto. Neste trabalho analisamos os efeitos de uma dieta rica em leucina sobre o metabolismo protéico em animais jovens prenhes portadores ou não do carcinossarcoma de Walker 256. Ratas Wistar foram distribuídas em grupos experimentais de acordo com a inoculação ou não do carcinossarcoma de Walker 256 e submetidas ou não a dieta rica em leucina. Após 20 dias de experimento foi realizado ensaios com o músculo esquelético (gastrocnêmio) a fim de elucidar o mecanismo de catabolismo tecidual que ocorre durante o processo de câncer-caquexia. Os grupos apresentaram aumento da taxa de proteólise muscular e redução da taxa de síntese protéica muscular. Porém, o grupo inoculado com tumor e tratado com dieta rica em leucina apresentou aumento da síntese protéica e menor espoliação de proteína muscular quando comparado com o grupo inoculado com tumor e não tratado com leucina na dieta. A suplementação de leucina na dieta, uma vez que este aminoácido é utilizado como fonte energética pelo músculo esquelético pode prevenir a depleção da carcaça, preservar a massa protéica corpórea e impedir o estado caquético do animal. Auxílio Financeiro: Fapesp (02/0644-7).
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INTRODUÇÃO
Apresentação geral
O câncer, proliferação e crescimento desordenado de células não controlados pelo organismo, pode causar danos ao tecido adjacente ou, até mesmo, à distância do tecido original. Multiplicando-se rapidamente, essas células tendem a ser muito agressivas e invasoras, determinando a formação de tumores ou neoplasias malignas.
A palavra câncer, por si só, abala psicologicamente os pacientes, culminando em desgaste emocional e, consequentemente, influenciando na qualidade de vida, que somados a outros fatores, como a falta ou redução das atividades físicas, a manifestação de dor e desordens metabólicas e bioquímicas corporais, abreviam a expectativa de vida dos pacientes.
A caquexia, presente no câncer, altera o metabolismo do organismo, principalmente, protéico e reduz a capacidade do organismo responder as terapias. A perda de peso e proteína corpórea contribuem para a manutenção do estado caquético.
Nas últimas décadas, os estudos realizados visam melhor conhecer os fatores biológicos, moleculares e genéticos do câncer. Intensificam-se aqueles relacionados às conseqüências e aos danos causados no organismo pelo crescimento neoplásico.
Tem-se observado importantes progressos na prevenção, diagnóstico e terapêutica do câncer. Recentemente, o redirecionamento dos padrões dietéticos vem ganhando adesão crescente para reduzir os efeitos negativos da caquexia e melhorar a qualidade de vida dos pacientes (Norman & Burtum, 2004) .
Em nossos estudos, temos verificado que o crescimento tumoral altera o metabolismo do organismo hospedeiro com redução do peso corpóreo e da ingestão alimentar. A redução do peso corpóreo, como conseqüência do decréscimo da proteína e gordura corpóreas, contribui para a instalação do estado caquético nesses animais. A evolução do tumor, também, causa redução do peso fetal e placentário, prejudicando assim a eficiência da troca de nutrientes entre mãe e feto (Ventrucci et al., 2001; Gomes-Marcondes, 2004).
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Demostramos, também, que a suplementação nutricional (aumento da ingestão de leucina) possui efeitos benéficos na composição corpórea química de ratas implantadas com tumor, preservando a massa corpórea magra e nitrogênio não-colágeno (Ventrucci et al., 2001). Apesar da redução do peso muscular, essas ratas inoculadas com tumor e tratadas com leucina na dieta, também, apresentam aumento do conteúdo protéico muscular (Ventrucci et al., 2002) e da quantidade de miosina em comparação ao grupo com tumor e não tratado com leucina (Ventrucci et al., 2004a).
Durante câncer-caquexia ocorre intensa mobilização de proteína corpórea e alterações no turnover protéico com redução da síntese e aumento da degradação. A dieta rica em leucina reduz a degradação de proteína muscular, provavelmente, inibindo a atividade do sistema ubiquitina-proteossomo, uma vez que a expressão das subunidades proteossômicas, 19S, 20S e 11S, está reduzida na associação câncer-leucina (Ventrucci et al., 2004a). Por outro lado, o aumento da síntese protéica muscular, observado por nós, no grupo de ratas com tumor e tratadas com leucina na dieta, associa-se à elevada expressão dos fatores de iniciação eIF2 e eIF5 e das proteínas S6 quinase e proteína quinase C (PKC) (Ventrucci et al., 2005). Desse modo, conhecer as vias e processos moleculares envolvidos na síntese e degradação protéica modulados pela leucina torna-se importante ferramenta para a prevenção da caquexia e, consequentemente, melhorar a resposta às terapias e qualidade de vida dos pacientes com câncer.
Câncer-caquexia
O câncer é considerado como a maior causa de morbidade e mortalidade ao longo da história humana e representa a segunda causa morte nos países desenvolvidos e também no Brasil, perdendo apenas para as doenças cardiovasculares (Buchalla et al., 2001). O crescimento de neoplasia maligna produz, no organismo hospedeiro, vários efeitos deletérios devido ao crescimento invasivo aos tecidos adjacentes e aparecimento de metástases (Svaninger et al., 1989). Dentre esses efeitos, podem ocorrer desordens nutricionais como desnutrição protéico-calórica e caquexia.
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função da intensa espoliação dos tecidos do hospedeiro, com intensa perda de peso, tecido adiposo e de massa muscular, alterando o metabolismo homeostático desses dois substratos, gordura e proteína (Inui, 1999). O desenvolvimento da caquexia possui impacto extremamente negativo aos pacientes com câncer (Tisdale, 2004). Durante a caquexia, ocorrem alterações na composição corpórea (Ventrucci et al., 2001) e aumento do catabolismo dos estoques energéticos, diminuindo a performance física (Tisdale, 2000).
Na vigência do câncer, evidenciam-se redução do apetite e, conseqüentemente, redução do peso corpóreo (Guaitani et al., 1983), com instalação do balanço nitrogenado negativo, diretamente relacionados à alta demanda de nitrogênio pelas células neoplásicas; o hospedeiro perde proteína tecidual e o nitrogênio protéico é seqüestrado pelo tumor (Argiles & Lopes Soriano, 1999). A perda de peso, decorrente da redução da gordura e de massa protéica corpórea nos pacientes com câncer-caquexia, é aumentada com a evolução do crescimento tumoral (Tisdale, 2000). Fatores catabólicos produzidos pelo tumor parecem ser os principais mediadores dessa perda de peso (Toledo & Gomes-Marcondes, 2004; Tisdale, 1997a,b), já que animais experimentais pair-fed (nutrição pareada aos animais com câncer) não apresentam a mesma perda de peso e de proteína muscular como os animais implantados com tumor (Tisdale, 2004; Ventrucci et al., 2001). O catabolismo protéico leva à astenia, caracterizada pela fadiga física e mental, comprometendo o sistema imune e aumentando a susceptibilidade as infecções (Tisdale, 2004). Pacientes que apresentam câncer-caquexia demonstram anormalidades no metabolismo protéico muscular esquelético, com conseqüente atrofia muscular, em função do desequilíbrio do turnover protéico corpóreo, reduzindo a síntese protéica e aumentando a degradação protéica (Inui, 1999; Tisdale, 1999 e 2004).
Leucina
Os aminoácidos assumem importante papel regulatório no metabolismo protéico, como os aminoácidos de cadeia ramificada (BCAA): leucina, valina e isoleucina (Nair, 1992). A leucina é importante combustível metabólico da musculatura esquelética e parece estimular a incorporação de aminoácidos nas
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proteínas e inibir a degradação protéica (Rennie & Tripton, 2000; Ventrucci et al. 2004b).
Pacientes com câncer caquexia apresentam significativa perda de peso corpóreo marcado pela redução de peso muscular, resultando na espoliação da proteína músculo esquelético (Acharyya et al., 2004; Ventrucci et al., 2002).
A proteólise muscular esquelética é regulada por vários mecanismos, sendo o sistema ubiquitina-proteossomo, via não-lisossomal, o principal responsável pela degradação protéica, quando a caquexia está estabelecida (Jagoe et al., 2002; Attaix et al., 1998). O sistema ubiquitina-proteossomo envolve a atividade das enzimas ubiquitina E1, E2 e E3 e do complexo proteossômico 26S que possui as subunidades 20S, 19S e 11S.
Alguns BCAAs possuem ações inibitórias sobre a proteólise da musculatura esquelética, podendo modular suas taxas em estados catabólicos, exercendo controle de feed back negativo (Argiles et al., 1996). Dietas contendo diferentes proporções de BCAAs apresentaram efeitos benéficos nas trocas metabólicas associadas à perda muscular (Louard et al., 1995; Busquets et al., 2000).
A leucina pode modular as taxas de proteólise (Argilés et al., 1996) e inibir a degradação de proteínas musculares esqueléticas (Busquets et al., 2000). Possui, ainda, efeitos benéficos no metabolismo protéico muscular revertendo a proteólise total muscular em ratos em jejum (Busquets et al., 2002)
A regulação da proteólise muscular é importante para a homeostasia energética, controle da massa muscular e crescimento corpóreo (Kettelhut et al., 1988).
Nas últimas 3 décadas, vários estudos sobre a tradução, função dos ribossomos e controle traducional foram realizados e muitas idéias indicam que há inter-relação entre o sistema de tradução e os fatores nutricionais, como a suplementação de aminoácidos.
O controle da tradução, amplamente discutido no contexto do músculo esquelético, é um processo de múltiplos passos, que envolve a disponibilidade dos fatores eucarióticos da iniciação (eIFs) e ativação da proteína ribossomal S6 quinase (S6K1) (Shah et al., 2000). O processo de síntese protéica inicia-se pela ligação do fator de iniciação eIF2 ao GTP e ao met-tRNAi, formando o complexo ternário
eIF2-5
GTP- met-tRNAi. Parte desse complexo ternário liga-se a subunidade ribossomal 40S, estabelecendo o complexo pré-iniciador 43S. Para que esse complexo possa reconhecer e ligar-se ao RNAm, é necessária a atuação do complexo eIF4F, formado pelo conjunto de proteínas: eIF4A, eIF4B, eIF4G e eIF4E (Anthony et al., 2001). O complexo eIF4F promove a ligação do complexo pré-iniciador 43S com a subunidade ribossomal 60S, estabelecendo a unidade 80S. O complexo eIF4F atua reconhecendo, desenrolando e guiando o RNAm durante a tradução. O fator eIF4E associado ao eIF4G identifica a região terminal 5´do RNAm na região m7GTP (Shah et al., 2000). Após o reconhecimento do códon AUG, inicia-se a tradução onde o movimento do complexo pelo RNAm é guiado pela atividade do eIF4A, estimulado pelo eIF4B.
O mecanismo pelo qual a atividade do complexo eIF4F regula a iniciação da tradução envolve a modulação e a disponibilidade de eIF4E. O fator eIF4E pode ligar-se à 4E-BP1 (repressora da tradução) e, enquanto estiver associado ao complexo 4E-BP1, o RNAm não pode ligar-se à unidade ribossomal 80S. A dissociação do complexo 4E-BP1 é regulada pela atividade da proteína mTOR (mammalian target of rapamycin), que fosforila a 4E-BP1 e, consequentemente, causa dissociação do complexo, liberando o eIF4E que se associa ao eIF4G (Kimball & Jefferson, 2004 a,b).
A atividade de várias moléculas envolvidas na maquinaria da tradução é modulada pela atividade da mTOR (Proud, 2002). Igualmente à 4E-BP1, a S6K1 é fosforilada e ativada pela mTOR, representando aumento da capacidade da célula em sintetizar proteínas (Proud, 2002; Kimball & Jefferson, 2004a). A mTOR serve como ponto de bifurcação na cascata da síntese protéica pela célula muscular esquelética sinalizada pela insulina e leucina (Pain, 1994). Aminoácidos podem ser reguladores positivos da mTOR. O mecanismo pelo qual os aminoácidos influenciam a síntese protéica ainda não está bem esclarecido, mas estudos sugerem que a síntese protéica aumenta quando ocorre elevada disponibilidade dos aminoácidos intracelulares (Proud, 2002).
Por outro lado, leucina pode estimular a síntese protéica através de seus metabólitos como o ácido cetoisocapróico (KIC). No processo catabólico da leucina, esta é transaminada em KIC, que por sua vez, pode ser o controlador das ações da
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leucina no metabolismo protéico (Nissen & Abumrad, 1997). O ácido cetoisocapróico é clivado, via mitocôndria, produzindo NADH e acetil-CoA que fornecerá ATP, através do ciclo de Krebs (Tokunaga et al., 2004).
Gravidez
Vários tipos de tumor têm sido descritos concomitante à gravidez, como câncer do cervix uterino, o mais comum (Germann et al., 2005), mama, melanona, linfoma e leucemia (Jacobs et al., 2004). Pesquisas mostram que a incidência de câncer de mama em mulheres gestantes é cerca de 2,8% (Gallenberg & LoprinzI, 1989). Parente et al. (1988) verificaram que 2 a 5% das pacientes com câncer de mama apresentavam gravidez concomitante, com ocorrência de morte fetal ou perinatal (King et al., 1985).
Estudos experimentais mostram que ratas portadoras de carcinossarcoma apresentam redução do peso fetal (Ventrucci et al., 2001), aumento da reabsorção (70%) e morte fetal (50%) (Gomes-Marcondes, 1998 e 2004; Cervello et al., 1992). Desta forma, o câncer, assim como outras patologias, pode interferir no processo gestacional (Gomes-Marcondes, 1994 e 2004). O diagnóstico e o tratamento terapêutico de pacientes grávidas, que apresentam câncer concomitante, são especialmente dificultados porque envolve dois pacientes, mãe e feto (Jacobs et al., 2004).
Na gravidez, ocorrem mudanças fisiológicas no organismo materno, preparando-o para suprir as necessidades do desenvolvimento e crescimento fetal, porém durante câncer-gravidez, o organismo materno também fornece nutrientes para o crescimento tumoral. O crescimento tumoral causa alterações no metabolismo materno bem como no metabolismo fetal, reduzindo a qualidade da gestação.
Durante a fase anabólica da gestação, ocorre aumento de tecido adiposo, glicogênio hepático e da massa corpórea magra (Carbo et al., 1998). Já na catabólica, ocorrem mobilização dos nutrientes e aumento do turnover protéico materno, que fornecem substratos ao crescimento fetal.
O crescimento tumoral por alterar o turnover protéico materno (Carbo et al., 1998) aumenta o catabolismo protéico muscular esquelético (Ventrucci et al.,
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2004a,b) levando à redução do aporte nutricional ao feto. Por outro lado, a produção de fatores catabólicos que induz a caquexia, também, pode promover prejuízos ao feto e, consequentemente, ao curso da gestação (Tisdale, 2000; Toledo & Gomes-Marcondes, 1999).
Nas últimas décadas, ocorreu melhora da taxa de sobrevivência de pacientes grávidas com câncer, devido ao uso combinado de cirurgia, radioterapia e quimioterapia. Porém, muitas drogas citotóxicas usadas na quimioterapia e, principalmente, radioterapia são potentes fatores teratogênicos e mutagênicos, podendo resultar em aborto e anormalidades fetais (Gbolade, 2000).
Avanços nos tratamentos terapêuticos para pacientes grávidas com câncer significam melhora da qualidade de sua vida reprodutiva (Laurence et al., 2004).
OBJETIVOS
Melhorar a resposta às terapias, intervenções cirúrgicas e sobrevida mostram-se imprescindíveis à condição câncer-caquexia. Portanto, esmostram-se trabalho teve como principal objetivo investigar o impacto da dieta rica em leucina nos processos de degradação e síntese protéica muscular esquelética e os fatores que possam minimizar e melhorar o estado caquético associado à suplementação nutricional.
8 RESULTADOS E DISCUSSÕES
Os resultados e discussões deste trabalho estão apresentados em dois artigos científicos.
O primeiro artigo corresponde aos resultados obtidos sobre síntese e degradação protéica e expressão das subunidades do proteossomo 26S do músculo esquelético, publicados na revista Endocrine-Related Cancer (Ventrucci et al., 2004). Neste trabalho descrevemos que a leucina pôde modular o metabolismo protéico muscular esquelético na vigência do câncer. O crescimento tumoral reduziu as taxas de síntese protéica e aumentou a proteólise muscular. Porém, a suplementação de leucina na dieta, no grupo com tumor, promoveu aumento da taxa de síntese protéica e menor espoliação da proteína muscular, provavelmente, em função da reduzida expressão das subunidades proteossômicas 19S, 20S e 11S, demonstrando a habilidade da leucina em alterar a atividade do sistema ubiquitina-proteossomo, envolvido no processo de degradação de proteínas musculares.
O segundo artigo refere-se aos resultados da incorporação de leucina e ácido cetoisocaproico (KIC), expressão dos fatores eucarióticos da iniciação (eIFs) e das proteína S6 quinase e proteína quinase C em músculo esquelético; submetidos à publicação na revista Endocrine-Related Cancer. Nesse trabalho observamos, também, que a leucina modulou os fatores envolvidos na sinalização celular para síntese protéica aumentando a expressão dos fatores eIF2α e eIF5, envolvidos no início do processo de tradução, e das proteínas S6K1, que sinaliza a tradução via mTOR, e PKC, que aumenta a captação de glicose via leucina, bem como prevenindo a redução da insulina plasmática, importante via de sinalização da síntese protéica.
9 PRIMEIRO ARTIGO CIENTÍFICO
Proteasome activity is altered in skeletal muscle tissue of tumour-bearing rats fed a leucine-rich diet.
Atividade proteossômica altera-se no músculo esquelético de ratos com tumor alimentados com dieta rica em leucina.
Resumo
Leucina pode modular o metabolismo protéico muscular aumentando a síntese protéica e reduzindo a proteólise. Neste estudo, investigamos os efeitos da leucina no sistema ubiquitina-proteossomo no músculo esquelético de ratas grávidas inoculadas com tumor e tratadas com dieta rica em leucina. Ratas Wistar grávidas alimentadas com dieta controle semi-purificada foram distribuídas em 3 grupos: C, controle; W, inoculado com tumor de Walker 256; P, pair fed. Três outros grupos tratados com dieta rica em leucina foram distribuídos em: L, leucina; WL inoculado com tumor de Walker 256 e PL pair fed. As ratas foram injetadas, no subcutâneo, com suspensão de células tumorais Walker 256. A síntese e degradação protéica foram medidas no músculo gastrocnêmio; a concentração de proteína total e a atividade das enzimas quimiotripsina e fosfatase alcalina, também, foram determinadas. Alíquotas de proteína muscular foram utilizadas para determinar a expressão de miosina de cadeia pesada (MHC) e das subunidades proteossômicas 20S, 19S MSSI ATPase e 11S. Embora o crescimento tumoral tenha reduzido a incorporação de [3H]-fenilalanina, a suplementação de leucina na dieta promoveu aumento da taxa de síntese protéica. Os grupos inoculados com tumor apresentaram aumento da proteólise muscular quando comparados aos respectivos grupos controles. Porém, a dieta rica em leucina causou menor degradação de proteína muscular no grupo WL do que no grupo W. Apenas o grupo W demonstrou redução significante (71%) do conteúdo de miosina. No grupo WL, houve redução da expressão das subunidades proteossômicas 20S e 11S (cerca de 32%), enquanto a expressão da subunidade 19S foi cerca de 3 vezes menor, quando comparado ao grupo W. Esses resultados indicam que a leucina pode estimular a síntese protéica e inibir a degradação protéica em ratas grávidas, provavelmente, pela modulação da atividade do sistema ubiquitina-proteossomo durante o crescimento tumoral.
Proteasome activity is altered in skeletal
muscle tissue of tumour-bearing rats fed
a leucine-rich diet
G Ventrucci, M A R Mello and M C C Gomes-Marcondes Nutrition and Cancer Research Laboratory, Department of Physiology and Biophysics, Institute of Biology, State University of Campinas (UNICAMP), Campinas, Sa˜o Paulo, Brazil, 13083-970
(Requests for offprints should be addressed to M Gomes-Marcondes; Email: cintgoma@unicamp.br)
Abstract
Leucine can modulate skeletal muscle metabolism by enhancing protein synthesis and decreasing proteolysis. In this study, we investigated the effects of leucine on the ubiquitin–proteasome system in skeletal muscle of pregnant tumour-bearing rats fed a leucine-rich diet. Pregnant Wistar rats were distributed into three groups that were fed a semi-purified control diet (C, control; W, Walker tumour-bearing; P, pair-fed) and three other groups of pregnant rats fed a semi-purified leucine-rich diet (L, leucine; WL, Walker tumour-bearing; PL, pair-fed). The tumour-bearing rats were injected subcutaneously with a suspension of Walker 256 tumour cells. Protein synthesis and degradation were measured in gastrocnemius muscle; the total protein content and tissue chymotrypsin-like and alkaline phosphatase enzyme activities were also determined. Muscle protein extracts were run on SDS-PAGE to assess the expression of the myosin heavy chain (MHC), 20S a proteasome subunit, 19S MSSI ATPase regulator subunit and 11S a subunit. Although tumour growth decreased the incorporation of [3H]-Phe, the concomitant feeding of a leucine-rich diet increased the rate of protein
synthesis. Muscle proteolysis in both tumour-bearing groups was increased more than in the respective control groups. Conversely, the leucine-rich diet caused less protein breakdown in the WL group than in the W group. Only the W group showed a significant reduction (71%) in the myosin content. In WL rats, the 20S proteasome content (32 kDa band) was reduced, while the expression of the 19S subunit was 3-fold less than in the W group and the 11S proteasome subunit reduced, to around 32% less than in the W group. These findings clearly indicate that leucine can stimulate protein synthesis and inhibit protein breakdown in pregnant rats, probably by modulating the activation of the ubiquitin–proteasome system during tumour growth.
Endocrine-Related Cancer (2004) 11 887–895
Introduction
Cachexia occurs in about one-half of all cancer patients (Tisdale 1997) and is the major cause of cancer morbidity and mortality. Cachexia is characterised by a progressive loss of body weight, especially in adipose and muscle tissues, which impairs normal functioning. The loss of skeletal muscle in most cachectic patients and animals involves a decrease in protein synthesis and increased protein degradation, as indicated by a variety of meta-bolic alterations (Ventrucci et al. 2002, Gomes-Marcondes et al. 2003).
Muscle proteolysis involves lysosomal and non-lyso-somal pathways. Non-lysonon-lyso-somal proteolysis is controlled
by the ubiquitin–proteasome system and involves intracel-lular and myofibrillar protein breakdown (Kee et al. 2002). The muscle ubiquitin pathway is a complex process activated during severe catabolism such as occurs in cancer cachexia (Attaix et al. 1999, Wray et al. 2002). This process involves multiple steps that are proteasome dependent, including protein ubiquitinization mediated by the ubiqui-tin enzymes E1, E2 and E3 and the 26S proteasome (Attaix et al.2001), which depends on ATP as an energy source. The 26S proteasome is a proteolytic complex that consists of a central core particle, the 20S proteasome subunit, and two regulatory complexes (19S and 11S) that are bound to the end of the 20S subunit (Glickman & Ciechanover 2002).
Endocrine-Related Cancer (2004) 11 887–895
Endocrine-Related Cancer (2004) 11 887–895 DOI:10.1677/erc.1.00828
Leucine plays an important role in skeletal muscle metabolism and can enhance protein synthesis and decrease proteolysis, independently of other branched-chain amino acids (BCAAs) such as isoleucine and valine (Carbo et al. 1996a, Anthony et al. 2001). Leucine regulates protein synthesis in skeletal muscle following food intake (Anthony et al. 2002b). A leucine-rich diet can improve the carcass nitrogen and lean body mass in rats with Walker 256 tumours and in mice with MAC 16 adenocarcinoma (Tisdale 2000, Ventrucci et al. 2001). The importance of insulin and amino acids, mainly BCAAs, in regulating protein synthesis has been studied in several situations (Fulks et al. 1975). The deleterious effects of tumours are more pronounced when associated with pregnancy since maternal nutrition supplies glucose and amino acids for tumour and foetal development (Carbo et al.1996a) and can result in muscle-wasting proteolysis. In this study, we investigated the effects of leucine on skeletal muscle protein synthesis and degradation, and the activity of the ubiquitin–proteasome system in pregnant tumour-bearing rats fed a leucine-rich diet.
Methods
Animals and diets
Young female Wistar rats (45 days old, n ¼ 60) were obtained from the animal facilities of the State University of Campinas, Sa˜o Paulo, Brazil. Female rats were housed overnight with adult males (four females:one male), according to harem methodology (Baker 1991), and the first day of pregnancy was determined based on the detection of sperm in the vaginal smear. All rats were housed in collective cages under standard conditions (22 2 8C, 12 light:12 h darkness cycle, with free access to water and food). Semi-purified diets were used and consisted of a balanced control diet (C; 18% protein, AIN-93G) and a leucine-rich diet (L; 15% protein with 3% leucine). The diets contained the same amount of carbohydrate (63%), fat (7%) and fibre (5%), in accordance with the AIN-93G (Reeves 1993). The corn standard and dextrin were supplied by Corn Products Brazil Ingredients, the vitamin mix was from DSM Nutritional Products (DSM Produtos Nutricionais Brasil Ltda), and the amino acids were from Ajinomoto Interamericana Ind. & Com. Ltda, Brazil.
The rats were randomly allocated to six groups. Three groups were fed the control diet: C, pregnant; W, pregnant tumour-bearing; P, pregnant pair-fed, this group received the same amount of food as ingested by the W group. Three other pregnant groups were fed with the leucine-rich diet: L, pregnant; WL, pregnant tumour-bearing; PL, pregnant pair-fed, this group received the same amount of food as ingested by the WL group.
Tumour implantation
The rats in groups W and WL received a subcutaneous injection of Walker 256 tumour cells (approximately 0:25 106cells in 0.5 ml saline solution) in the right flank
immediately after the confirmation of pregnancy. The other pregnant groups without tumours (controls) received a single injection of 0.5 ml 0.9% (w/v) NaCl in the same region as a control manipulation. All of the groups were monitored for 20 days after tumour implantation. The general guidelines UKCCCR (United Kingdom Co-ordinating Committee on Cancer Research 1988) for animal welfare were followed, and the protocols were approved by the institutional Committee for Ethics in Animal Research (CEEA-IB/UNICAMP, protocol 217-5).
Experimental procedures
Protein synthesis was assayed in right gastrocnemius muscles, which were weighed and placed in Krebs– Henseleit bicarbonate (KHB) buffer (110 mM NaCl, 25 mM NaHCO3, 3.4 mM KCl, 1 mM CaCl2, 1 mM
MgSO4and 1 mM KH2PO4, pH 7.4) supplemented with
5.5 mM glucose and 0.01% (w/v) albumin. The muscles were pre-incubated for 30 min at 37 8C with continuous gassing (95% O2–5% CO2) and shaking, as described by
Vary et al. (1998). After this period, new KHB buffer supplemented with 5 mCiL-[3H]phenylalanine/ml
(Amer-sham) was added and the incubation continued for a further 2 h. At the end of this period, the muscles were homogenised in 10% trichloroacetic acid (TCA, 1:3 w/v), centrifuged at 10 000 g for 15 min at 4 8C, and the pellet then suspended in 1 M NaOH and incubated at 40 8C for 30 min. Aliquots of this mixture were used to measure the total protein content (Bradford 1976) and to quantify the radioactivity based on liquid scintillation counting of b emissions. The rate of protein synthesis was calculated by the amount of radioactive phenylalanine incorporated in a 2 h period, and was expressed as nanomoles of [3H]-Phe
per microgram of muscle protein (Vary et al. 1998). Protein degradation was assessed using left gastro-cnemius muscles which were excised and placed in RPMI 1640 medium and pre-incubated for 30 min under the same conditions of temperature and gas as described above (Vary et al. 1998). After the initial incubation, the solutions were replaced by KHB buffer supplemented with cycloheximide (130 mg/ml) followed by a 2 h incuba-tion. At the end of this period, the muscles were dried, weighed and frozen in liquid nitrogen. The muscles and incubation medium were stored at 80 8C until analysis. The rate of protein degradation was determined as nanomoles of tyrosine released per microgram of muscle protein per hour, based on fluorometric assay as described by Waalkes & Udenfriend (1957).
Ventrucci et al.: A leucine-rich diet affects muscle proteasome activity
Enzymatic activities
The proteins of the gastrocnemius muscle were homo-genised in homogenising buffer (HB) (20 mM Tris, 1 mM dithiothreitol, 2 mM ATP and 5 mM MgCl2) and
centri-fuged at 15 000 g for 15 min at 4 8C. The resulting super-natant was analysed for total protein content (Bradford 1976) and chymotrypsin-like and alkaline phosphatase activities. Chymotrypsin-like activity was determined using the fluorogenic substrate succinyl-Leu-Leu-Val-Try-7-amino-4-methylcoumarin (Suc LLVY-AMC; 0.167 mg/ml in 100 mM Tris–HCl, pH 7.4; excitation 360 nm, emission 460 nm). The activity was expressed as units of fluorescence per microgram of protein, as a percentage of the control group. Alkaline phosphatase activity was measured using 37 mM p-nitrophenyl phosphate (PNPP) as substrate, and activity was expressed in nanomoles of nitrophenol formed per microgram of muscle protein (Martins et al. 2001).
Myosin and proteasome system
Skeletal muscle proteins were resolved by SDS-PAGE on 12% gels followed by Western blotting. The content of myosin heavy chain (MHC) isoforms was assessed using antibody against MHC at a dilution of 1:250 (Novocastra, Newcastle, UK), followed by detection with a secondary anti-mouse horse radish peroxidase (HRP) antibody (Dako, Carpinteria, USA). The proteasome subunits were analysed by probing 0.45 mm enhanced chemical luminescence (ECL) nylon membranes with antibodies against the 20S a proteasome subunit, the 19S MSSI ATPase regulator subunit and the 11S a subunit (all from Affinity, Newcastle, UK, and diluted 1:1500) followed by detection with the rat anti-mouse secondary HRP-labelled antibody. Actin was used as the loading control, after probing the mouse actin antibody. Images of the gels were captured (FTI 500Image Master VDS, Pharmacia Biotech) and densitometric analyses of the bands were done with Gel Pro Analyser software (Media Cybernetics, Silver Spring, MD, USA)
Statistical analysis
The results are expressed as the means S.E.M.Statistical comparisons were done with one-way ANOVA (Gad & Weil 1994) followed by Bonferroni’s test for comparison among groups (Graph Pad Prism software, v3.00 for Windows 98, USA). Statistical significance was consid-ered as a P value below 5%.
Results
Protein synthesis
The rate of incorporation of [3H]-Phe by the gastrocnemius muscle, indicative of protein synthesis, was higher in the
L group (around 23.4%) than in the corresponding control group, C (Fig. 1A). Tumour growth reduced this parameter in both tumour-bearing groups (W and WL), with a decrease in [3H]-Phe incorporation by about 47 and 40% respectively, compared with the respective C and L groups. Whereas tumour growth induced severe damage in both pregnant groups (W and WL), the leucine-rich diet fed concomitantly with tumour growth resulted in increased protein synthesis in the WL group (around 1.4-fold higher than in the W group). These results suggest that leucine probably had a protective effect on skeletal muscle
C W P L WL PL 1.0 3.5 6.0 b c c c d a A e ni n al al y n e h P /µl o m n() h/ ni et or p g C W P L WL PL 0.05 0.10 0.15 0.20 a c a c b c B e ni s or y T / sti n u e c n e c s er o ulf yr ar ti br a( ) h/ ni et or p µg
Figure 1 Effects of a leucine-rich diet on protein synthesis (A) and degradation (B) in gastrocnemius muscle from pregnant tumour-bearing rats. Right gastrocnemius muscles from all experimental groups were incubated with [3H]-Phe for 2 h and
protein synthesis was then assessed based on the incorporation of radiolabelled amino acid. The contralateral gastrocnemius muscles were similarly incubated for 2 h with cycloheximide to measure protein breakdown based on the release of tyrosine. At least eight to ten rats were used per group. Abbreviations: C, control; W, tumour-bearing rats; P, pair-fed tumour-bearing rats; L, leucine-rich diet group; WL, tumour-bearing rats receiving leucine-rich diet; PL, pair-fed tumour-bearing group fed with a leucine rich-diet. Columns with different letters above are significantly different (P < 0:05).
Endocrine-Related Cancer (2004) 11 887–895
metabolism. Anthony et al. (2002b) showed that the oral administration of leucine increased muscle protein synth-esis in rats. Pair-fed nutrition does not decrease the rate of protein synthesis, in contrast to the decrease caused by neoplasic growth (Smith & Tisdale 1993). Indeed as shown here, the incorporation of phenylalanine was not reduced in the P and PL groups as compared with the tumour-bearing groups.
Protein degradation
Muscle proteolysis, represented by the release of tyrosine from gastrocnemius muscles, was compared between control and leucine-rich diet groups (Fig. 1B). There was no difference in tyrosine release from skeletal muscle in the C and L groups. However, during tumour growth, there was an imbalance between the rates of protein synthesis and degradation, especially in the W groups. Body weight loss and muscle fatigue are common in cancer cachexia and in women with gynaecological cancers (Olt 2003). In recent studies, we observed a body weight loss and a decrease in total body protein in pregnant rats with Walker 256 tumour (Gomes-Mar-condes et al. 1998, Ventrucci et al. 2001). As shown in Fig. 1B, both tumour-bearing groups showed increased tyrosine release, around 33.5% higher than in the respective control groups, indicating intense muscle protein mobilisation during tumour growth. The tumour-bearing rats fed a leucine-rich diet (WL) showed significantly reduced tyrosine release rates, by around 11% when compared with the W group. An increase in tyrosine release was also verified in the pair-fed groups, P (18%) and PL (21%), when compared with the respective control groups (C and L). However, in the pair-fed situation, the protein breakdown was less intense than in the W and WL groups.
Enzyme activities
The chymotrypsin-like activity (Fig. 2A) was increased in all experimental groups compared with group C, although the activity was lower in the WL group (around 17.8% less) compared with the W group. In the leucine pair-fed group, muscle chymotrypsin-like activity was also similar to the L group. In addition, as presented in Fig. 2B, there was a significant increase in muscle alkaline phosphatase activity only in the pair-fed groups, P and PL, compared with the C and L groups respectively.
MHC in gastrocnemius muscle
Cancer cachexia wastes whole host tissue by specifically reducing the protein carcass mass. Both of the tumour-bearing groups (W and WL) presented a reduction in
muscle myosin content (Fig. 3). The reduction in group W was significantly greater (71%) than in the corresponding control group (C). On the other hand, there was a significant increase in the myosin content (47%) of gastrocnemius muscle in WL rats in comparison to the W group. Comparatively, as seen in Fig. 3, the muscle myosin content was preserved in both of the pair-fed groups.
Ubiquitin–proteasome system
Muscle proteolysis in cancer cachexia results from high stimulation of the ubiquitin–proteasome system. Feeding a leucine-rich diet to tumour-bearing rats significantly improved the host muscle mass by altering the expression of the proteasome subunits. Both bands of 20S
protea-C W P L WL PL 0.0 2.5 5.0 7.5 a b b b b b A yti vi t c a ni s p yr t o m y h c el c s u M e c n e c s er o ulf yr ar ti br a( /µ sti n u) h/ ni et or p g C W P L WL PL 0.00 0.25 0.50 0.75 a b a a a b B
Alkaline phosphatase activity
(µ l/µ o m) h/ ni et or p g
Figure 2 Effects of a leucine-rich diet on muscle enzyme activities in tumour-bearing rats. Muscle chymotrypsin-like and alkaline phosphatase activities were measured in gastrocne-mius muscle homogenate. Eight to ten rats per group. For abbreviations, see legend to Fig. 1. The columns are the means S.E.M.for eight to ten rats per group. Columns with different letters above are significantly different (P < 0:05).
Ventrucci et al.: A leucine-rich diet affects muscle proteasome activity
some subunit (Fig. 4) were significantly increased in the W rats when compared with the other groups. Although the expression levels of the two bands of 20S proteasome slightly increased in groups fed a leucine-rich diet, in the WL group the 20S subunit expression (32 kDa band) was around 32% less than in the W group (W, 0.230 0.012 arbitary units; WL, 0.156 0.0154; P < 0:05). There was no significant difference in levels of the 20S subunits between the pair-fed groups. The expression of the 19S subunit (Fig. 5) in the WL group was 3-fold lower than in the W group (W, 0.417 0.017; WL, 0.137 0.010 arbitary units; P < 0:05), with no difference in the pair-fed groups. A slight increase in the 11S proteasome subunit (Fig. 6) was observed in the W group compared with group C, but was significantly higher (32% in the W group) only when compared with the pair-fed (P and PL) and the leucine-rich diet groups (W, 0.511 0.068; WL, 0.348 0.057 arbitary units; P < 0:05).
Discussion
The present findings clearly indicate that a leucine-rich diet altered the activation of the ubiquitin–proteasome system during tumour growth. Amino acids, especially
C C W W P P L L WL WL PL Myosin (220 kDa) A C W P L WL PL 0.0 0.5 1.0 1.5 a a c c b c B t n et n o c ni s o y m el c s u M ) yti s n e d yr ar ti br a( Actin (42 kDa)
Figure 3 Effects of a leucine-rich diet on muscle MHC content in tumour-bearing rats. (A) Western blot image of MHC and actin. Actin was used as a loading control. The Western blots are representative data of the best assay from each group (minimum of eight rats per group). (B) Arbitrary densitometric values of myosin expression are analysed for eight individual Western blots. Gastrocnemius muscle homogenates were loaded into SDS-PAGE gels (5mg protein/well), later transferred to nylon membranes and blotted with an MHC antibody (diluted 1:250). For details see Methods. For abbreviations see the legend to Fig. 1. The columns are the means S.E.M.for eight to ten rats per group. The expression of actin (42 kDa band) showed no difference. Different letters above each column indicate statistical difference ðP < 0:05Þ.
A C C W W P P L L WL WL PL PL 20S subunit (33 kDa) (32 kDa) C W P L WL PL 0.0 0.1 0.2 0.3 B a b b a,b a,b a,b e m o s a et or p S 0 2 el c s u M ti n u b u s yr ar ti br a a D k 3 3( ) yti s n e d C W P L WL PL 0.0 0.1 0.2 0.3 a b a,b a,b a a,b e m o s a et or p S 0 2 el c s u M ti n u b u s yr ar ti br a a D k 2 3( ) yti s n e d Actin (42 kDa)
Figure 4 Effects of a leucine-rich diet on expression of the 20S asubunit of the 26S proteasome in tumour-bearing rats. (A) Western blot image of 20S subunit and actin. Actin was used as a loading control. Western blots are representative data of the best assay from each group (minimum of eight rats per group). (B) Arbitrary densitometric values of the expression of the 20S subunit (33 and 32 kDa bands) are analysed for eight individual Western blots. Gastrocnemius muscle homogenates were loaded into SDS-PAGE gels (5 mg protein/well), later trans-ferred to nylon membranes and blotted with a 20S a subunit proteasome antibody (diluted 1:1500). For details see Methods. For abbreviations see legend to Fig. 1. The columns are the means S.E.M.for eight to ten rats per group. The expression of actin (42 kDa band) showed no differences. Columns with different letters above are significantly different ðP < 0:05Þ.
Endocrine-Related Cancer (2004) 11 887–895
BCAAs, can improve and enhance the rates of protein synthesis in skeletal muscle (Kettelhut et al. 1988, Yoshizawa et al. 1998, Anthony et al. 2002b).
In previous studies, we observed benefits of a leucine-rich diet on carcass mass during Walker tumour growth (Ventrucci et al. 2001, Gomes-Marcondes et al. 2003). The present results now show a positive effect of a leucine-rich diet on muscle protein turnover.
Tumour growth causes intense tissue protein turnover by reducing protein synthesis and/or by increasing protein catabolism (Smith & Tisdale 1993, Tessitore et al. 1993). Since a reduction in caloric intake does not decrease skeletal muscle mass, as shown in pair-fed rats of the present study and verified by other studies in animals with cachectic tumours (Smith & Tisdale 1993), the cause of the increase in muscle wasting associated with cancer
cachexia remains unknown (Tisdale 2002, 2003). Although Walker 256 tumour growth decreases skeletal muscle and whole maternal body weight, feeding a leucine-rich diet prevented the intense reduction, as previously reported (Ventrucci et al. 2001, Gomes-Marcondes et al. 2003). As shown here, 20 days after tumour implantation, a leucine-rich diet significantly enhanced protein synthesis (Fig. 1A). As reviewed by Anthony et al. (2001), BCAAs can regulate protein synthesis in skeletal muscle. Protein synthesis is stimu-lated in starved rats, especially after the infusion of amino acids by total parenteral nutrition (Svanberg et al. 1998). These authors also noted that the protein synthesis was enhanced in myofibrillar L6 cell culture, following the addition of amino acids plus insulin-like growth factor (IGF-I) to the culture medium.
C W P L WL PL 0.0 0.1 0.2 0.3 0.4 0.5 a a a b a a B ti n u b u s e m o s a et or p S 9 1 el c s u M ) yti s n e d yt ar ti br a( C W P L WL PL 19S subunit (48 kDa) Actin (42 kDa) A
Figure 5 Effects of a leucine-rich diet on expression of the 19S MSSI ATPase subunit of the 26S proteasome in tumour-bearing groups. (A) Western blot image is representative of the best assay from all experimental groups. (B) Arbitrary densitometric values of eight individual Western blots. Gastrocnemius muscle homogenates were loaded into SDS-PAGE gels (5 mg protein/ well), later transferred to nylon membranes and blotted with a 19S MSSI ATPase subunit proteasome antibody (diluted 1:1500). For details see Methods. For abbreviations see legend to Fig. 1. The columns are the meansS.E.M.for eight to ten rats per group. Columns with different letters above are significantly different ðP < 0:05Þ. C C W W P P L L WL WL PL PL 11S subunit (30 kDa) A C W P L WL PL 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 a,b a a b a a B ti n u b u s e m o s a et or p S 1 1 el c s u M ) yti s n e d yr ar ti br a( Actin (42 kDa)
Figure 6 Effects of a leucine-rich diet on expression of the 11S asubunit of the 26S proteasome in tumour-bearing groups. (A) Western blot image of the 11S a subunit and actin. Actin was used as a loading control. The Western blot is the represen-tative data of the best assay from each group (minimum of eight rats per group). (B) Arbitrary densitometric analysis of 11S a expression values for eight individual Western blots. Gastro-cnemius muscle homogenates were loaded into SDS-PAGE gels (5 mg protein/well), later transferred to nylon membranes and blotted with an 11S a subunit proteasome antibody (diluted 1:500). For details see Methods. For the abbreviations see legend to Fig. 1. The expression of actin (42 kDa band) showed no differences. Columns with different letters above are significantly different ðP < 0:05Þ.
Ventrucci et al.: A leucine-rich diet affects muscle proteasome activity
The acute administration of leucine stimulates protein synthesis in various tissues of fasted rat (Vary et al. 1999, Anthony et al. 2002b, Lynch et al. 2002). The administra-tion of leucine by gavage to food-deprived rats elevated the rates of protein synthesis in skeletal muscle (Anthony et al. 2002a). In rats perfused with amino acid solutions, Vary et al.(1999) verified that amino acid supplementation with leucine increased the total protein synthesis in gastro-cnemius muscle compared with values obtained with a leucine-poor solution.
Our results support the hypothesis that leucine alone has beneficial effects on skeletal muscle metabolism in tumour-bearing animals, and this was confirmed by the increase in the myosin content (Fig. 3) of the gastro-cnemius muscle in WL animals.
In agreement with the idea that leucine alone can improve protein metabolism in tumour-bearing rats, the ingestion of a leucine-rich diet during pregnancy partly inhibited the proteolysis seen in the WL group. Busquets et al. (2000) showed that leucine (10 mM solution) inhibited muscle proteolysis in the extensor digitalis and soleus muscles after incubation with a solution to activate the proteolytic system. Recently, Busquets et al. (2002) reported that the addition of leucine to the incubation medium had no effect on total muscle proteolysis in muscles from Yoshida tumour-bearing rats. In contrast to Busquets et al. (2002), we found that leucine alone stimulated protein synthesis, and inhibited the protein breakdown, probably by reducing the activation of the ubiquitin–proteasome system.
The ATPase subunit of the 19S complex (MSSI) and the 11S subunit associate with the 20S proteasome (Coux et al. 1996), and these two regulatory complexes can be responsible for modulated activation of proteolysis; in addition, the two 20S subunits, a and b, can be regulated independently (Attaix et al. 1998, Tanahashi et al. 1999). An increased expression of the MSSI could facilitate the rapid proteolysis of muscles in Walker tumour-bearing rats since the MSSI unit provides energy for the breakdown of ubiquitinylated proteins by the 26S proteasome (Coux et al.1996). The enhanced proteolysis seen in cancer-wasting conditions is ATP dependent (Attaix et al. 1997, 1998) since there is an increase in the expression of several ATPases, including MSSI (Dawson et al. 1995, Fujita et al. 1996, Combaret et al. 1999). Since ATPases provide energy for assembly of the 26S proteasome and for the breakdown of ubiquitin conjugates (Attaix et al. 1998, Combaret et al. 1999), the reduction seen here in the 19S proteasome MSSI subunit parallel to an increase in the myosin content suggested that the ubiquitin–proteasome system may be regulated by BCAAs, such as leucine. Larbaud et al. (1996) showed that ubiquitin was also regulated by insulin, with a marked decrease in ubiquitin expression in skeletal muscle
after treatment with insulin. Insulin can improve the host carcass protein turnover (Curi et al. 1995, Fernandes et al. 1996a,b), and Anthony et al. (2002a) have observed simultaneous stimulatory effects of insulin and leucine on protein turnover in skeletal muscle.
In an extensive review, Coux et al. (1996) suggested that the 11S regulator subunit associates with the 20S proteasome to stimulate peptidase activity and could be involved in the final breakdown of peptides derived from protein degradation. Although there was no significant difference in 11S expression between the W and C groups, this expression was lower in the WL group, suggesting that supplementation with leucine conferred some advantage.
Muscle protein mobilisation can be an adaptable process to several conditions such as pregnancy in order to provide amino acids and an adequate nutritional supply for foetal growth (Carbo et al. 1998); it can also adapt to diseases, to provide amino acids for acute energy metabolism cost and other essential processes for organism survival. On the other hand, tumour growth can affect protein turnover and lead to ineffective amino acid transport between the mother and foetus (Carbo et al. 1996b). Since adequate maternal nutrition is funda-mental for foetal welfare and placental development (Godfrey 2002), protein catabolism intensifies in late pregnancy to provide enough nutritional support for foetal growth (King 2000). Conversely, progressive tumour development leads to foetal weight loss (Ven-trucci et al. 2001, 2002) and compromises the placental– foetal exchange (Toledo & Gomes-Marcondes 1999), mainly through the direct and/or indirect effects of cytokines produced by the tumour and/or host cells (Toledo & Gomes-Marcondes 2004a,b). Other studies have shown that cytokines (Kettelhut et al. 1988) and, more recently, a proteolysis-inducing factor (Gomes-Marcondes et al. 2002, Hasselgren et al. 2002, Tisdale 2003) cause intense muscle wasting by activation of the ubiquitin–proteasome system.
The precise mechanism by which leucine and/or BCAAs can induce protein synthesis and/or inhibit protein breakdown needs to be better understood. Investigations are currently underway to address these important questions. In practical terms, our results suggest that the ingestion of a leucine-rich diet could be beneficial in reducing the muscle wasting associated with cancer cachexia.
Acknowledgements
This study was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (Fapesp)
Endocrine-Related Cancer (2004) 11 887–895
(96/9463-9466, 01/02135-02133, 02/04464-04467), Con-selho Nacional de Desenvolvimento Cientı´fico e Tecno-lo´gico (CNPq) (521048/95-98, 522755/96-98, 350047/03-00), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Fundo de Apoio ao Ensino e a` Pesquisa (FAEP-UNICAMP).
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Endocrine-Related Cancer (2004) 11 887–895
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19 SEGUNDO ARTIGO CIENTÍFICO
Effects of leucine rich diet on translation eukaryotic initiation factors in skeletal muscle of tumour-bearing pregnant rats
Efeito da dieta rica em leucina sobre os fatores eucarióticos da iniciação da tradução em músculo esquelético de ratas prenhes com tumor.
Resumo
Câncer-caquexia induz uma variedade de desordens metabólicas no metabolismo protéico que envolve redução da síntese protéica e aumento da degradação protéica. Insulina, outros hormônios e nutrientes, como os aminoácidos de cadeia ramificada, especialmente leucina, estimulam a síntese protéica e modulam a atividade de fatores eucarióticos da iniciação no mecanismo de síntese protéica. Ratas grávidas foram implantadas ou não com tumor de Walker 256 e distribuídas em 6 grupos: três grupos foram tratados com dieta controle (18% caseína): C controle, W inoculada com tumor e P pair fed, que recebeu a mesma quantidade de dieta ingerida pelo grupo W; outros três grupos foram tratados com dieta rica em leucina (3% leucina e 15% caseína): L leucina, WL inoculado tumor e PL pair fed, que recebeu a mesma quantidade de dieta ingerida pelo grupo WL. O grupo WL apresentou aumento da incorporação de leucina e KIC nas células do músculo gastrocnêmio comparado ao grupo W. A dieta rica em leucina preveniu a redução da insulina plasmática. No grupo inoculado com tumor e tratado com dieta rica em leucina, houve aumento da expressão dos fatores de iniciação, eIF2α e eIF5 cerca de 35%, e dos fatores eIF4E e eIF4G, cerda de 17% e 20% maior, quando comparado ao grupo W; o grupo tratado com leucina apresentou aumento da expressão de S6K1 e PKC, 50% e 56%, respectivamente. A dieta rica em leucina estimulou a síntese protéica muscular esquelética no grupo inoculado com tumor, aumentando vários fatores de sinalização, provavelmente, interferindo na síntese protéica através dos fatores eIFs e/ou ativando a via da S6 quinase.
Effects of leucine rich diet on translation eukaryotic initiation factors in skeletal 1
muscle of tumour-bearing pregnant rats 2
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Ventrucci G, Mello MAR, Gomes-Marcondes MCC
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Nutrition and Cancer Research Laboratory. Departamento de Fisiologia e Biofísica,
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Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas,
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São Paulo, Brazil, 13083-970.
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Correspondence to M.C.C. Gomes-Marcondes: cintgoma@unicamp.br
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Key words: leucine, tumour Walker 256, skeletal muscle, eukaryotic initiation factors,
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protein kinase C.
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Running title: Leucine diet on eukaryotic initiation factors
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Abstract: 15
Cancer-cachexia induces a variety of metabolic disorders in protein metabolism that
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involves a decrease in protein synthesis and an increase in protein degradation.
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Insulin, other hormones and nutrients, such as branched chain amino acids, especially
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leucine, stimulate protein synthesis and module the activity of a number of
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translational initiation factors in protein synthesis mechanism. Pregnant rats were
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implanted or not with Walker 256 tumour and distributed into six groups: three groups
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were fed with the control diet (18% casein): C pregnant control, W tumour-bearing,
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and P pair-fed, which received the same amount of food as ingested by the W group;
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three other pregnant groups were fed with the leucine-rich diet (3% leucine plus 15%
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casein): L pregnant leucine, WL tumour-bearing, and PL pair-fed, which received the
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same amount of food as ingested by the WL group. The gastrocnemius muscle of WL
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groups increased the leucine and KIC incorporation to protein compared to W rats.
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Leucine-rich diet prevent the plasma insulin reduction. The translational initiation
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factors were improved when the tumour-bearing rats were fed a leucine-rich diet:
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eIF2α and eIF5 expressions were around 35% increased; eIF4E and eIF4G were
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around 17% and 20% higher then W group; and S6K1 and PKC were 50% and 56%
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enhanced, respectively. The leucine-rich diet increased protein synthesis in skeletal
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muscle in tumour-bearing rats and enhanced many signalling factors, probably
interfering in protein synthesis by the eIF factors and/or activating the S6kinase 34 pathways. 35 36 37 Introduction 38
Cancer-cachexia induces a variety of metabolic disorders in the whole body,
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including marked weight loss, especially in adipose and muscle tissues
(Gomes-40
Marcondes et al. 2003). Skeletal muscle loss causes alteration in protein metabolism
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that involves a decrease in protein synthesis and an increase in protein degradation
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(Ventrucci et al. 2004, Vary & Kimball 2000). Insulin, other hormones and nutrients,
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such as branched chain amino acids (Nishitani et al. 2004) stimulate protein synthesis.
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Leucine, a branched-chain amino acid, stimulates the muscle protein synthesis and
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modules the activity of a number of proteins involved in the control of mRNA
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translation (Kimball et al. 2000, Beugnet et al. 2003).
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Leucine may stimulate the protein synthesis by itself acting directly action
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and/or by its metabolite, the α-ketoisocaproic acid (KIC) (Nissen & Abumrad 1997).
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On the other hand, protein synthesis is regulated by interactions between mRNA,
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tRNA and eukaryotic initiation factors (eIFs). Leucine can stimulate the translation
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initiation by the mammalian target of rapamycin (mTOR) and independently (Fedele et
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al. 2001, Holecek et al. 2001, Proud 2002). However, leucine play stimulatory effects 53
on glucose uptake by protein kinase C (PKC) messenger, differently from insulin
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effects on glucose uptake that occurs via protein kinase B (PKB) (Nishitani et al.
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2002).
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The aim of this study was to verify the effects of a leucine rich diet on the
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skeletal muscle of tumour-bearing rats, since tumour growth induces an increase of
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protein degradation and a reduction in protein synthesis and these two process were
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altered when the tumour-bearing rats were fed a leucine rich diet.
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Material and Methods 62
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Animals and diets 64
Young female Wistar rats (45 days old, n=60) were obtained from the animal
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facilities of State University of Campinas, São Paulo, Brazil. The rats were housed
